System and method for selective measurement of microbial activity in a water sample

Abstract

A system and method for selectively determining the microbial activity of peroxide sensitive microbes in a water supply using the differential concentration of stabilized hydrogen peroxide in a demand test. The system and method measures the concentration of live peroxide sensitive microbes in a water system using hydrogen peroxide demand as a proxy for live microbial concentration. Calculation of a microbial activity score provides information on the risk of infection by peroxide sensitive microbes in a water system.

Description

SYSTEM AND METHOD FOR SELECTIVE MEASUREMENT OF MICROBIAL ACTIVITY IN A WATER SAMPLECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to United States provisional patent application US63 / 571,510 filed 29 March 2024, which is hereby incorporated by reference herein in its entirety.FIELD OF THE INVENTION

[0002] The present invention pertains to a system and method for determining deleterious microbial activity in a water supply. The system and method measure the concentration of live microbes in a water system using hydrogen peroxide demand as a proxy for live peroxide sensitive microbe concentration.BACKGROUND

[0003] Hydrogen peroxide (H2O2) can be used in water distribution systems for primary and secondary disinfection and treatment of pathogenic microbes without the formation of halogenated disinfection by-products that result from the chemical reaction of halogen disinfectants, such as those containing chlorine, with organic materials. Opportunistic pathogens in water distribution systems pose a significant risk to human health and chronic use of traditional chemical disinfectants that leads to the formation of disinfection by-products can be challenging in both non-circulating and recirculating water systems. In building hot water lines or circulating hot water, for example, conditions in the water distribution system can be ideal for microbiological growth, as water already treated with primary disinfection may have insufficient residual disinfectant to control pathogen growth, which increases risk of user exposure and infection. Purpose-built water systems such as cooling towers, heating ventilation and air conditioning (HVAC) systems, evaporative condensers, pools, and spas, can also have locations of stagnant water in plumbing systems that can provide favourable conditions for growth of opportunistic pathogens if disinfection is not maintained in proper control.

[0004] Risk assessment and continuous monitoring of municipal and premise water supplies is critical to ensuring water safety and providing risk mitigation of opportunistic pathogens in water distribution systems. In water distribution systems such as in aquaculture, agriculture, and recreational water systems, obtaining an accurate measurement of the concentration of livedeleterious microbes in the system can assist with reducing the use of chemical disinfectants to the level required for eliminating live pathogens, eliminating its overuse. Right-amount use of chemical disinfectants limits the production of disinfection by-products and controls the development of antibiotic resistance in the form of super bugs such as resistant environmental strains of Escherichia coli and mycobacteria. Similarly, understanding microbial load in a water supply can also reduce energy use through reduction of ultraviolet treatment, heat treatment, and pump use. Emerging opportunistic pathogens with antibiotic resistance further undermines public water health. Climate change, extreme weather events such as flooding, and pandemics also require greater water resilience.

[0005] Hydrogen peroxide is secreted by plants at various stages in a plant life cycle and is used in regulation of plant metabolism as well as cellular signalling in response to environmental stress. During environmental stress in plants, the concentration of intracellular and extracellular hydrogen peroxide increases. Many plants also have peroxidases, which are enzymes that react with hydrogen peroxide and catalyze the degradation of hydrogen peroxide. As such, plants have a protective mechanism against peroxides in the environment. The use of hydrogen peroxide in plant irrigation systems can therefore be very useful in controlling deleterious microbes in agricultural water systems as plants are not sensitive to low concentrations of peroxide, however opportunistic microbes generally are sensitive to peroxide. In single use in circulating agricultural water systems such as those used in many greenhouses, deleterious plant microbes can bloom on plant nutrients and runoff, causing early rot and / or damage to plants and food products, putting a high disinfection demand on the water system. Poor water quality in agricultural growing systems also inhibits root health, increases the risk of plant disease, and decreases crop weight at harvest time. Measuring and controlling microbiological growth, and particularly monitoring and control of pathogens, is critical for maintaining health in many types of water systems.

[0006] Enzymes are complex biological molecules that are synthesized by living cells with intact membranes and are essential catalysts in metabolic processes for growth and replication, including biotransformation and biodegradation reactions. Live cells have enzymes such as peroxidases and catalases that react with peroxides in solution to cause peroxide decomposition or degradation. In a water system comprising viable microbial cells, measuring the rate ofperoxide decomposition reflects the level of enzymatic activity from live or enzymatically active microbes in the system that are reactive with peroxide at the concentration of peroxide in solution. Hydrogen peroxide demand can be directly correlated with enzymatic activity, specifically catalase and peroxidase, of bacteria and eukaryotic microbes in the water sample that are reactive with peroxide at the test concentration. In one study in an aquaculture system, Lars- Flemming Pedersen et al. (Assessment of microbial activity in water based on hydrogen peroxide decomposition rates, Aquacultural Engineering. Volume 85, 2019, pages 9-14, ISSN 0144-8609) describe an assay for assessment of microbial activity in a stagnant water sample by observing the degradation rate of a hydrogen peroxide (H2O2) over time using spectrophotometric determination of concentration of H2O2 over a sixty-minute time period. The assay described by Pederson et al. illustrates how peroxide decomposition can be used as a probe to assess microbial enzymatic activity in a stagnant water sample after a single peroxide dosing. However, Pederson et al. does not address the contribution of peroxide sensitive microbes compared to peroxide- nonreactive microbes in the water sample.

[0007] Many circulating water systems, particularly in North America, depend on chlorine as a primary disinfectant, with control systems that measure the concentration of circulating chlorine species to determine whether the water is safe and has sufficiently low levels of microbes. However, active chlorine is susceptible to heat shock and can bind with metals such as copper, silver, and iron, making it less effective at disinfection. This makes chlorine species concentration a poor indicator for microbial growth in the water system as variations in pH, temperature, total organic carbon (TOC), metal concentration, and other factors, can significantly affect the concentration of chlorine species in the water system. Primary ultraviolet disinfection can be highly effective at water disinfection, at the treatment point, however most water systems consist of a complex microbiome, and biofilms can accumulate downstream of the disinfecting lamps and in peripheral locations in the water system. Left unchecked, pathogenic microbes may be harboured and thrive throughout the water system. A healthy microbiome in a well managed water system can be good for water integrity and provide control of deleterious pathogens.

[0008] In one method for maintaining disinfectant levels in a water system, United States patent US 11,827,533 B2 to Giguere et al. describes obtaining a water sample and adding achlorine-containing material to the water sample in the presence of an oxidation reduction potential (ORP) measurement device to determine the disinfectant composition of the water.

[0009] The traditional method of measuring microbial activity in a water sample is culture testing, however culture results take time to acquire, require sterile conditions, are generally non- quantitative, and are prone to error. Molecular specific methods including live / dead staining and microbe specific tracking, optionally coupled with high throughput flow cytometry, are costly and require specialized facilities and relatively complex sample preparation. There remains a need for a system and method for selectively measuring microbial activity in a water supply in the presence of a mixed microbiome.

[0010] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.SUMMARY OF THE INVENTION

[0011] An object of the present invention is to provide a method of measuring live microbial concentration in a water sample using demand of stabilized hydrogen peroxide as a proxy for concentration of live peroxide sensitive microbes in the water sample.

[0012] In an aspect there is provided a method for detecting hydrogen peroxide sensitive microbes in a water system comprising: taking a water sample from the water system; adding a known amount of stabilized hydrogen peroxide (SHP) to the water sample to provide an initial concentration of hydrogen peroxide in the water sample; waiting a delay time and; calculating a microbial activity of peroxide sensitive microbes in the water sample by colorimetrically determining a concentration of hydrogen peroxide after the delay time.

[0013] In another aspect there is provided a method for detecting hydrogen peroxide sensitive microbes in a water system comprising: obtaining a water sample from the water system; adding an initial known amount of stabilized hydrogen peroxide (SHP) to the water sample to provide an initial concentration of hydrogen peroxide in the water sample; waiting a delay time; and calculating a microbial activity of peroxide-sensitive microbes in the water sample by colorimetrically determining a concentration of hydrogen peroxide after the delay time.

[0014] In an embodiment, the stabilized hydrogen peroxide is hydrogen peroxide stabilized with silver.

[0015] In another embodiment, the silver comprises one or more of silver ions, oligodynamic silver, silver nanoparticles, and silver colloid.

[0016] In another embodiment, the initial amount of stabilized hydrogen peroxide provides a hydrogen peroxide concentration in the water sample of between about 1-125 ppm.

[0017] In another embodiment, the water sample is taken at multiple locations in the water system.

[0018] In another embodiment, the water system is a potable water system, agricultural water system, horticultural water system, aquaculture system, or recreational water system.

[0019] In another embodiment, the microbial activity score (MAS) in the water system based on the hydrogen peroxide degradation rate over time is calculated as:where [SHP] is the concentration of stabilized hydrogen peroxide in the water sample.

[0020] In another embodiment, the calculation of MAS further comprises adjusting the MAS calculation based on one or more of quality control reagent check, MAS risk (short), MAS resilience (long), reaction time, dilution, concentration of Pseudomonas-type organisms, concentration of Bacillus- ype organisms, concentration of Lactobacillus-type organisms, concentration of Trichoderma-type organisms, concentration of iron or iron-type metals, concentration of total organic carbon (TOC), alkalinity, calcium hardness, concentration of one or more species of chlorine-type sanitizers, conductivity, total dissolved solids (TDS), pH, turbidity, and water system temperature.

[0021] In another embodiment, the method further comprises adjusting a disinfection regimen of the water system when the microbial activity score of peroxide sensitive microbes in the water sample is greater than a safety threshold for microbial control.

[0022] In another embodiment, adjusting the disinfection regimen comprises one or more of dosing the water system with a chemical disinfectant, increasing the pump rate of a disinfectant pump into the water system, increasing ultraviolet treatment in the water system, shocking the water system with a chemical disinfectant, increasing ozone production in the water system,adding beneficial microbes to the water system, and performing a maintenance operation on the water system.

[0023] In another embodiment, the disinfection regimen lowers the microbial load of peroxide sensitive microbes in the water system to within the safety threshold.

[0024] In another aspect there is provided a system for detecting hydrogen peroxide sensitive microbes in a water system comprising: a water sample reservoir; a peroxide reagent reservoir comprising stabilized hydrogen peroxide; a mixing reservoir fluidly connected to the water sample supply and the peroxide reagent reservoir for receiving and mixing a water sample and stabilized hydrogen peroxide to provide a reacted water sample; a peroxide dye reservoir comprising peroxide dye; a colorimeter for receiving the peroxide dye and the reacted water sample and performing a colorimetric measurement to determine a concentration of hydrogen peroxide in the reacted water sample; and a water health processor for receiving colorimetry data from the colorimeter.

[0025] In another aspect there is provided a system for detecting hydrogen peroxide sensitive microbes in a water system comprising: a water sample supply for receiving a water sample from the water system; a peroxide reagent reservoir comprising stabilized hydrogen peroxide; a mixing reservoir fluidly connected to the water sample supply and the peroxide reagent reservoir for receiving and mixing a water sample and stabilized hydrogen peroxide to provide a reacted water sample; a peroxide dye reservoir comprising peroxide dye; a colorimeter for receiving the peroxide dye and the reacted water sample and performing a colorimetric measurement to determine a concentration of hydrogen peroxide in the reacted water sample; and a water health processor for receiving colorimetric measurements from the colorimeter and determining a microbial activity score for the water system based on the colorimetric measurements.

[0026] In an embodiment the system further comprises one or more of sediment filter, dilution reagent reservoir, pump, flow meter, sanitizing rinse reservoir, and matrix reagent reservoir.

[0027] In another embodiment the system further comprises more than one water sample reservoir and more than one mixing reservoir.

[0028] In another embodiment, the stabilized hydrogen peroxide is silver-stabilized hydrogen peroxide.

[0029] In another embodiment, the water sample supply is in line in a water system.In another embodiment, the water health processor is connected to one or more water system sensor selected from the group consisting of a turbidity sensor, pH probe, temperature probe, conductivity probe, dissolved oxygen probe, flowmeter, water usage sensor, water waste sensor, pump flow rate sensor, pump speed sensor, and pump variable frequency drive sensor.

[0030] In another embodiment, the water health processor further comprises one or more of an artificial intelligence engine and machine learning analytics engine.

[0031] In another embodiment, the water health processor provides a process control signal to effect an action on a controllable variable in the water system.

[0032] In another embodiment, the controllable variable comprises flowrate, pressure differential, filter operation and backwashing, water level, chemical level, flow rate, pump rate, and water temperature.

[0033] Embodiments of the present invention as recited herein may be combined in any combination or permutation.BRIEF DESCRIPTION OF THE FIGURES

[0034] For a better understanding of the present invention, as well as other aspects and further features thereof, reference is made to the following description which is to be used in conjunction with the accompanying drawings, where:

[0035] Figure 1 is a schematic diagram of a system for measuring microbial load in a water sample;

[0036] Figure 2 illustrates a general method for measuring microbial load in a water sample;

[0037] Figure 3 is a flowchart illustrating a method for measuring microbial load in a water sample;

[0038] Figure 4 is a schematic diagram of a system for measuring microbial load in multiple water samples;

[0039] Figure 5 is a schematic diagram of a system for monitoring and controlling microbial load in water;

[0040] Figure 6 is a graph of the degradation of hydrogen peroxide over time caused by Pseudomonas chlororaphis

[0041] Figure 7 is a graph of the change in MAS over time caused by Lactobacillus complex at a test sample concentration of 1 million cfu / mL;

[0042] Figure 8A is a graph of the change in MAS over time caused by Lactobacillus complex at a test sample concentration of 10,000 cfu / mL;

[0043] Figure 8B is a graph of the change in MAS over time caused by Bacillus spectrum,'

[0044] Figure 9 is a graph of the change in MAS over time caused by Trichoderma complex;

[0045] Figure 10 is a graph illustrating the effect of iron on the degradation of hydrogen peroxide over time;

[0046] Figure 11 is a graph of the effect of total organic carbon (TOC) on the degradation of hydrogen peroxide over time in a water sample;

[0047] Figure 12 is a graph of the multiple-component factor effect on the degradation of hydrogen peroxide over time;

[0048] Figure 13 is a graph of the measurement of Lactobacillus complex on microbial activity score (MAS) vs time in the presence of E. coll,

[0049] Figure 14 is a graph of the measurement of MAS on a mixed sample containing Trichoderma complex and coliforms;

[0050] Figure 15 is a graph of the measurement of MAS on a sample containing only coliforms;

[0051] Figure 16 is a graph illustrating microbial risk levels compared with water temperature in a greenhouse water circulation system;

[0052] Figure 17 shows part of a graphical user interface with results from a water health index system reporting health insights based on MAS;

[0053] Figure 18 is a graph of iron vs. concentration of stabilized hydrogen peroxide in solution;

[0054] Figure 19A illustrates a graphical user interface reporting a high risk condition for a hospital shower;

[0055] Figure 19B illustrates the MAS data showing the MASa (short) data over time compared to cATP data over time indicating a microbe hot spot at the hospital shower;

[0056] Figure 20 illustrates MAS data in a propagation greenhouse;

[0057] Figure 21A illustrates a graphical user interface after detection of high MAS and before remediation in a propagation greenhouse;

[0058] Figure 21B illustrates a graphical user interface after detection of high MAS and after remediation in a propagation greenhouse;

[0059] Figure 22 illustrates MAS data in a greenhouse silo;

[0060] Figure 23 illustrates MAS data at a transplant table in a greenhouse;

[0061] Figure 24A illustrates a front isometric view of an example system for measurement of microbial activity in a water sample in a benchtop apparatus;

[0062] Figure 24B illustrates a rear isometric view of the example system in a benchtop apparatus;

[0063] Figure 24C illustrates a top view of the example system in a benchtop apparatus;

[0064] Figure 24D illustrates a front isometric view of the example system in a benchtop apparatus with an open door or removable panel; and

[0065] Figure 24E illustrates a front isometric view of the example system in a benchtop apparatus with a removable or sliding front panel showing the mixing reservoirs.DETAILED DESCRIPTION OF THE INVENTION

[0066] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0067] As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise.

[0068] The term “comprise” and any of its derivatives (e.g. comprises, comprising) as used in this specification is to be taken to be inclusive of features to which it refers, and is not meant to exclude the presence of any additional features unless otherwise stated or implied. The term “comprising” as used herein will also be understood to mean that the list following is non- exhaustive and may or may not include any other additional suitable items, for example one or more further feature(s), component(s) and / or element(s) as appropriate.

[0069] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. A composition, device, article, system, use, or method described herein as comprising certain elements and / or steps may also, in certain embodiments consist essentially of those elements and / or steps, and in other embodiments consist of those elements and / or steps, whether or not these embodiments are specifically referred to.

[0070] As used herein, the term “about” refers to an approximately + / -10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to. The recitation of ranges herein is intended to convey both the ranges and individual values falling within the ranges, to the same place value as the numerals used to denote the range, unless otherwise indicated herein.

[0071] The use of any examples or exemplary language, e.g. “such as”, “exemplary embodiment”, “illustrative embodiment” and “for example” is intended to illustrate or denote aspects, embodiments, variations, elements or features relating to the invention and not intended to limit the scope of the invention.

[0072] As used herein, the terms “connect” and “connected” refer to any direct or indirect physical association between elements or features of the present disclosure. Accordingly, these terms may be understood to denote elements or features that are partly or completely contained within one another, attached, coupled, disposed on, joined together, in communication with, fluidly connected with, operatively associated with, etc., even if there are other elements or features intervening between the elements or features described as being connected.

[0073] The term “stabilized hydrogen peroxide” (SHP) as used herein refers to a solution of hydrogen peroxide in water comprising a stabilizer. SHP with a stabilizer remains stable longer in highly diluted form and retains residual levels of active peroxide at low concentrations in different kinds of water matrixes. Unlike conventional hydrogen peroxide, the reaction of stabilized hydrogen peroxide is controlled and degradation is more gradual, enabling enhanced stability and efficacy against target micro-organisms and protection of water or wet surface integrity, in cold and warm water conditions at a wide pH range. A variety of stabilizers can be used such as, for example, low concentrations of metal ions such as copper or silver, and organic stabilizers such as, for example tartrate salts, citrate salts, and chelating agents such as aminophosphonates and organic compounds with primary amine groups. The stabilizer delays the reaction of hydrogen peroxide and prolongs its stability in water, thereby allowing the peroxide to react with microorganisms in the present application.

[0074] The term “silver stabilized hydrogen peroxide” (SHP-Ag) refers to a stabilized hydrogen peroxide solution in water in which the stabilizer hydrogen peroxide is based on silver. The stabilizer can include one or more forms of silver, including, for example, silver ions, oligodynamic silver, silver nanoparticles, and silver colloid. The silver is present in minute concentrations, for example in compositions comprising about 5-50% hydrogen peroxide, there is about 0.003 and 0.030% of silver by weight in volume (mg / L). SHP stabilized with silver (SHP-Ag) displays significant biocidal activity towards gram-positive and gram-negative bacteria that are capable of producing significant levels of peroxide-neutralizing catalase. The silver component of SHP-Ag helps target the hydrogen peroxide to the bacterial cell surface through electrostatic interactions, thereby creating high local concentrations of peroxide. It has been proposed that the addition of silver helps to stabilize the highly labile peroxide, thereby enhancing the residual levels of peroxide in solution. SHP-Ag has also been demonstrated to associate with the bacterial cell surface to a greater extent than peroxide alone, and this surface interaction is believed to contribute to the enhanced stability and effectiveness of SHP-Ag in relation to peroxide while enabling targeting, by mechanism of electrostatic interaction, of the cell wall of target micro-organisms, and controlled reactions with and oxidation of organic material and target organisms. (Martin NL, Bass P, Liss SN (2015) Antibacterial Properties and Mechanism of Activity of a Novel Silver- Stabilized Hydrogen Peroxide. PLOS ONE 10(7)).

[0075] As used herein, the term “water integrity” is used to refer to the quality of water in a water system, and is used primarily herein with respect to disinfection control of the deleterious microbial load in the water system. In water systems with high integrity the microbial load of deleterious microorganisms will be low and manageable with disinfection treatment. Water systems with low integrity have been seen to have wider fluctuations in microbial load, and growth can be unpredictable and result in water with higher than acceptable deleterious microbial load.

[0076] As used herein, the term “microbial load” refers to the number, mass, amount, or concentration of microbiological organisms in a water system. Increases in microbial load in awater system can occur for a number of reasons, and have been found to occur, for example, during heavy rainfall, snowmelt, flooding, rising groundwater events, higher temperature weather, and runoff events. During these events water turbidity, pH, and nitrate values can differ significant from the values obtained for regular samples, and bacteriological presence in the water can increase considerably during extreme runoff events. Microbial load is traditionally measured in colony forming units per mL (cfu / mL) using an incubation test in bacterial growth medium. It is understood that not all microbiological organisms measured as a part of the microbial load are pathogenic or deleterious, and that some microorganisms are probiotic or beneficial microbes. However overall higher microbial load is generally used as a measure of all microbial growth, where high microbial growth is understood to be detrimental to a safe water supply. If large numbers of non-pathogenic coliforms, for example, are found in water, there is a high probability that other pathogenic bacteria or organisms, such as giardia and cryptosporidium, may be present. Microbial load can be tested in a variety of ways, including but not limited to polymerase chain reaction (PCR), inoculative culture testing, and circulating cellular ATP (cATP) measurement. Microbial loading can result from bacteria, yeast, mould, fungi, and viruses, and the term “microbial load” is regarded as encompassing a quantitative value that captures all living biologicals in a water system or sample.

[0077] As used herein, the term “disinfection demand” refers to the amount of disinfectant required or minimum inhibitory concentration in the water system to maintain a healthy water supply with low microbial load. Disinfection demand can be measured in amount of disinfectant required over time, or can be understood by the disinfection setpoint, which is the concentration of disinfectant required by the system to maintain water integrity. The term “demand” as specifically used herein with reference to testing for the presence of peroxide sensitive microorganisms refers to the amount of hydrogen peroxide reduction in a water sample after exposure of the hydrogen peroxide to a condition that reduces the amount of hydrogen peroxide in the water sample.

[0078] As used herein, the acronym “cATP” refers to circulating cellular ATP, which is a measurement of circulating ATP in the water sample. ATP is an energy molecule produced by all living organisms, and can be used as an indicator of microbial activity.

[0079] The term “microbial activity score” or “MAS” refers herein to a measurement of the microbial demand for hydrogen peroxide in what is called a “demand test” or “MAS test”. The hydrogen peroxide demand from microbiology is the result of peroxide sensitive microbes in a water sample. The determination of MAS takes into account the degradation of hydrogen peroxide in a control water sample over time to determine the amount of peroxide degradation caused by microbes in a test water sample. This calculated MAS value is expressed as a percentage score, with a lower percentage score indicating a lesser amount microbiology in the sample water, and a higher percentage indicating a greater amount of microbiology in the sample water.

[0080] Herein is described a method and system for measuring the microbial activity of live peroxide sensitive microbes in a complex water system. The present system and method measures the concentration of live microbes in a water sample that are peroxide sensitive and calculates the potential risk in a water system using a hydrogen peroxide-based reagent degradation as a proxy for distinguishing live pathogens amid a potentially beneficial mixed microbiome in a water system. The present system and method can also take into account other interference parameters, such as, for example, the presence of metal ions and dissolved and suspended organic matter, which can be factored into a microbial activity calculation to provide a more accurate measurement of the amount of peroxide sensitive microbes in the water sample.

[0081] Many beneficial microbes either secrete hydrogen peroxide as a protective mechanism, or are not peroxide sensitive, meaning that they do not substantially break down or react with hydrogen peroxide in water at low peroxide concentrations. Examples of these beneficial microbes include, for example, lactic acid bacteria such as Lactobacillus plantarum, Bacillus pumilus and Bacillus subtilis, and fungal biocontrol agent Trichoderma harzianum, which have been shown to have protective effects on plant growth. Furthermore, hydrogen peroxide has long been used as a chemical disinfectant for potable water systems and demonstrated effectiveness at ridding water of potential pathogens that are harmful to people and animals.

[0082] It has been found that stabilized hydrogen peroxide, and specifically silver-stabilized hydrogen peroxide, can be used as an interrogative agent to probe for the presence of peroxidesensitive microorganisms in a water sample. Gram positive, gram negative, facultative anaerobic, and aerobic potentially beneficial bacteria, as well as beneficial fungi were tested. Possible mechanisms of action for the non-reactivity of beneficial microorganisms in the presence of hydrogen peroxide have been found. These relate to, for example, energy metabolism flexibility, gene expression peroxidase switches, internal hydrogen peroxide generation, as well as external mechanistic features such as genus competition, and immune supportive signalling to host organisms such as plants.

[0083] The present method and system can be used for detecting microbial risk in a mixed microbiome water system, with computation and analytical engines to calculate, predict and provide remediative feedback for peroxide sensitive microbial control. This method and system provides a rapid, inexpensive, and useful microbial risk analysis in real-world water systems with mixed microbiomes and functions as a peroxide resistance test in a population of mixed microorganisms. Using stabilized hydrogen peroxide demand as a proxy for calculating the quantity or concentration of live peroxide sensitive microbes in the water system, water in a distribution system can be tested off-line or in a standalone system, and testing at regular intervals provides a real time indication of the activity of live peroxide sensitive microbes in the water system.

[0084] Quantification of microbial activity in the water system can be used to inform downstream adjustments and process control to the disinfection regimen and water system as a whole. In particular, disinfection protocols, disinfectant amounts, location of application, cleaning regimens, and general process control conditions can all be adjusted in real time in response to accurate measurements of peroxide sensitive microbe concentration in the water system. Positive outcomes from ongoing microbial testing are mitigation of microbial risk, optimizing treatment regimens, and supporting greater microbial resilience and balance in a water system. Testing the concentration of live peroxide sensitive microbes in a water sample in real-time can also be used as a part of water integrity and water health measurement and reporting, providing a cultivation-free, reliable method to quantify the concentration of viable microbiology in water systems.

[0085] Figure l is a schematic diagram of a system for measuring microbial load in a water sample. Measurement system 10 for measuring peroxide degradation in a water systemcomprises water sample supply, which can be a water sample reservoir 12 where water is received from a water system, and a peroxide reagent reservoir 14 for storing stabilized hydrogen peroxide. The measurement system can be used as an off-line system or a standalone system for receiving water from a water system that may have suspended microbes for testing. Dilution reagent reservoir 16 stores clean water that will neither react with the sample water or the stabilized hydrogen peroxide. The dilution water can be used to dilute the sample water when the sample water has a high microbial count that makes concentration quantitation challenging in the colorimeter, or when the water sample has high turbidity which may impede light propagation in a colorimetric assay. The dilution water can also be used to dilute the sample water if the sample water has significant color or if the colorimeter cannot be calibrated or zeroed as required to measure peroxide concentration in the water sample. The colorimeter has a measurement cell to which is added an aliquot of sample water to be measured, such as between 7.5 mL and 15 mL of water sample. An optional sediment filter 22 can be used if the sample water is turbid or contains sediment to filter out sediment but retain the resident microbes in the water sample for measurement. Mixing reservoir 24 is connected to the water sample reservoir 12, peroxide reagent reservoir 14, and dilution reagent reservoir 16, and receives the sample water, stabilized hydrogen peroxide, and any diluent, and provides a chamber in which the contents of the water samples can react over time. Test aliquots of water reacted with peroxide can be removed from the mixing reservoir over time to provide a time course of the concentration of peroxide in the sample water over time in order to calculate the rate of reaction of the microbes resident in the sample water with the stabilized peroxide.

[0086] When a water sample has reacted with SHP and is ready for processing, an aliquot of the reacted water sample is sent to the colorimeter 26 along with a dye that reacts with peroxide from peroxide dye reservoir 18, where the dye is detectable in a colorimetric assay in colorimeter 26. Matrix reagent reservoir 20 is used when there remains significant yellow color in the sample, that with dilution prevents the colorimeter from being able to calibrate to zero.Formation of a reaction product between the peroxide dye and any peroxide remaining in the reacted water sample is proportional to the amount of hydrogen peroxide in the water sample. In one embodiment the peroxide dye is potassium bis (oxalato) oxotitanate (IV), optionally mixed with one or more additives, for example, one or more chelator such as EDTA di-sodium saltdihydrate, one or more surfactants such as polyoxyethylene (23) lauryl ether, and a pH balanced buffer. The wavelength for the emitter and detector in the colorimeter is selected to correspond with the selected reagent. Preferably the light emitter comprises a light emitting diode (LED) light which emits at a wavelength in the absorption band of the peroxide dye. In one reagent and emitter combination, the selected wavelength of the emitter is 470 nm. In a preferable embodiment the reagent comprises potassium bis (oxalato) oxotitanate (IV) DI, EDTA di-sodium salt dihydrate and polyoxyethylene (23) lauryl ether mixed in a solvent. In one example, the amount of peroxide dye reagent addition per sample is between about 0.25 mL and 0.5 mL. A measurement cycle for a single water sample can comprise between 8 and 16 samples per measurement cycle for each sample aliquot. A measurement cycle for a water sample can include both MASa Risk (0-60 mins) and MASa Resilience (20-24 hrs). A MASa curve timing engine can further trigger a colorimeter measurement cycle according to previously obtained MASa results. The colorimeter also can also be subject to a rinse, such as 15mL or other setpoint of dilution reagent, between each colorimetric measurement. A sanitizing rinse cycle of the system can also be timed between Ihr to 24hrs based on the water matrix. Quantification of the reaction product of hydrogen peroxide with the peroxide dye is done and converted to a peroxide concentration based on a standard curve. In one embodiment, the standard curve comprises data points from 0 ppm to 150 ppm. Additional details on peroxide concentration measurement using a colorimeter can be found in the inventors’ United States Patent 9,835,601 B2 granted on December 5, 2017, incorporated herein by reference.

[0087] Data collected by the colorimeter on the concentration of peroxide measured in the reacted water sample is then sent to a water health processor 28. Water health processor 28 is connected to a computer and a memory, such as random access memory (RAM) or other dynamic storage device (e.g. dynamic RAM (DRAM), static RAM (SRAM), and synchronous DRAM (SDRAM)) for storing information which may include temporary variables or other intermediate information and instructions to be executed by water health processor 28. Memory structures such as registers for storing temporary variables or other intermediate information may also be included in water health processor 28. The computer may also include a cache memory coupled to processor 28 for storing and providing faster access to frequently used data. The computer may further include a read-only memory (ROM) or other read-only storage that mayinclude but is not limited to programmable ROM (PROM), erasable ROM (EROM), and electrically erasable ROM for storing read-only information and instructions for the processor 28.

[0088] The water health processor 28 may receive data from a plurality of water system sensors 40a, 40b and 40c which are used to collect information about controllable variables and conditions in a given water system. Examples of controllable variables that may collected for a water system include but are not limited to water temperature, filter operation, backwashing, and water level sensors. For example, in recreational water systems such as spas and pools, information about pool turnover rate, number of guests, and deck temperature may be collected can be used to control controllable variables in the water system to provide water stability with varying water conditions. The water system sensors 40 may also be used to collect relevant information about the water system and may include, for example, one or more temperature sensor, pH probe, turbidity sensor, and flowmeter. Collection of additional data enables adjustments to be made to the monitoring of a water system for more accurate results, as well as application of feedback control. For example, the water health processor 28 may receive data indicating that the turbidity of water may be high and exceed a given threshold. As a result, the water health processor 28 may be programmed to pass a sample of water to be measured that exceeds this threshold through an optional sediment filter 22 in order to obtain a more accurate MAS reading. Alternatively, the water health processor 28 may, upon determining a MAS score, adjust any one of a number of controllable variables based on data obtain from one of a plurality of water system sensors 40 to adjust system control parameters in the water system as a whole. For example, a high MAS reading may prompt the water health processor 28 in a recreational water system such as a pool to increase the pool turnover rate or increase the level of disinfectant in order to improve water quality.

[0089] Figure 2 illustrates a general method for measuring microbial load 100 as a microbial activity score (MAS) of peroxide sensitive microbes in a water sample. To determine MAS in a water sample, a water sample to be measured is first obtained from a water system or water reservoir 102. The water system can be, for example, a potable water system, agricultural water system, horticultural water system, aquaculture system, recreational water system, or natural or environmental water system. The water sample is received into a mixing reservoir to which isadded a known amount of stabilized hydrogen peroxide (SHP) 104. If there is need to dilute the water sample, the water sample can be diluted with clean, non-reactive water 106. Some reasons that the water sample may need to be diluted are if the sample has high turbidity, high microbiological load, or if the water sample contains a coloured component that may interfere with colorimetry. Additionally, if there are chlorine disinfectant species present in the water sample the chlorine needs to be removed prior to incubation. One method for removing chlorine species is by measuring the amount of chlorine species in the water sample and adding 0.7 ppm of peroxide to neutralize each 1 ppm of reactive chlorine. When the peroxide is mixed with the water sample, the peroxide will react with any component in the water sample suceptible to reaction. Stabilized peroxide, in particular silver-stabilized peroxide, is generally stable in water over the time courses used in the present method, and therefore any reduction in concentration of peroxide will be due to the presence of peroxide sensitive microbes in the water sample. The water sample and peroxide mixture is then allowed to sit for a reaction time for components of water sample to react with the peroxide 108.

[0090] Introducing a known amount of hydrogen peroxide to the water sample containing microbes having metabolic enzymes that degrade the peroxide provides data on the concentration of peroxide sensitive microbes in the water sample. The difference between the added concentration of peroxide to the water sample and the concentration of peroxide after time t is referred to as the peroxide demand, which can be understood as the reduction of concentration of peroxide over the time t. Measurement of the concentration of peroxide in water sample at time t 110 provides a quantitation of the demand. This difference or demand of hydrogen peroxide is directly correlated to the concentration of peroxide sensitive microbes in the water sample, meaning that this analytical technique only measures the concentration of live microbes capable of peroxide degradation in the sample. As shown herein, peroxide concentration is also limitedly or not significantly affected by, for example, total organic carbon (TOC) concentration, the concentration of metals at reasonable concentrations that would be found in water samples for testing, or other contaminants that can potentially give inaccurate results. As such, peroxide demand can provide a real-time measure of the concentration of live peroxide sensitive microbes in the water system.

[0091] In a water sample containing a microbial population, the peroxide degradation activity, also referred to herein as the microbial activity, can be assigned a microbial activity score (MAS) based on the reduction of the amount of hydrogen peroxide in the water sample over time. Once the demand has been measured quantitatively, the microbial activity score in the water sample for time t can be calculated. Calculation of MAS at time 1 112 is done by measuring the amount of hydrogen peroxide in a water sample at time t in the presence of microbes, taking into account the degradation of peroxide under control conditions. In the present system and method, MAS can be measured in terms of concentration of SHP in the water sample after delay time t as:where:[SHP]t=0is the expected concentration of hydrogen peroxide in the water sample prior to reaction with components in the water sample, taking into account the amount of stabilized hydrogen peroxide added, the sample water volume, and any reduction in concentration of hydrogen peroxide observed in a control condition; and[SHP]t=tis the colorimetrically measured concentration of hydrogen peroxide in the water sample at time t.

[0092] The MAS percent (MAS%) can also be expressed as a number between 0 and 100, where a water sample having MAS near 0 is considered to be water having low peroxide sensitive microbial concentration, and a water sample having MAS nearer 1 is considered to be water having high microbial concentration. MAS can be expressed as MAS % as: 100

[0093] If it is desirable to calculate a peroxide decomposition rate over time, more than one aliquot of reacted water sample can be taken from the mixing reservoir at various times and demand measured at each different time point to provide a degradation rate curve. In a complex water sample, the calculation of MAS can take into account other factors such as the effects of dissolved and suspended organic material and metals, and the contribution of beneficialmicroorganisms in the sample to provide an indication of the microbial load of peroxide sensitive microbes in the water sample. Increased demand for or rate of degradation of hydrogen peroxide can serve as an early warning sign for changes in water quality and an early indicator of microbial population growth or a potential microbial bloom in the water system; earlier than other microbial testing methods. By continuous or periodic measuring of the peroxide demand in a water system over time the change in demand can signal a rising level of microbial activity in the water system, which may indicate the onset of a deleterious microbiological bloom. This early indicator can direct the disinfection regimen in a water system to respond by quickly treating the water supply to avoid the microbial bloom expected in the absence of appropriate additional treatment or slipping below the minimum inhibitory concentration of the disinfection regiment in the water system.

[0094] Based on the MAS calculated, the water health processor may be used to prescribe an action to reduce the concentration of live peroxide sensitive microbes 114. The presently described method and system can be implemented in directional as well as circulating water systems and may also be adapted to automated water sampling and measurement processes in a variety of industries including but not limited to plant agriculture, animal husbandry, premise plumbing, municipal water systems, food processing facilities, and recreational water systems. The measurement of hydrogen peroxide demand in a water sample of unknown microbiology composition and concentration can serve as a proxy for microbiological load or concentration of peroxide sensitive microbes in the system. Automated sampling and MAS scoring of water may occur as often as, for example, every 90 seconds, every 2 minutes, every 5 minutes, every 10 minutes, every 30 minutes, every hour, or at another interval. Frequent measurements in sensitive water systems are advantageous for detecting the rapid changes in microbial load that may occur in a flowing water system and to prevent high load events which can be harmful to humans, animals, and plants.

[0095] The automated water monitoring system can either feed MAS data into a disinfection regimen, which is programmed to maintain a set-point of a disinfectant level in the system via, for example, a proportional-integral-derivative controller (PID controller) loop, or query the water continuously in a sample stream. In this way, a reliable disinfection control regimen using any method of disinfection can be achieved by using hydrogen peroxide demand as a proxyconcentration of live peroxide sensitive microbiology in the water sample. Data arising from this quantification of live microbiology is used to inform the disinfection regimen and adjust the level of disinfectant or other water treatment components or controllable variables in accordance with the calculated level of live microbiology in the system and adjust the water system conditions to maintain disinfection control. Adjustments to the disinfection process can be initiated when the system detects a rise in peroxide sensitive microbiology in the water system. In response, an increase in disinfectant addition to the system, and optionally specifically at particular locations where the rise in microbiology concentration has been detected, can assist with maintaining disinfection control by way of controlling controllable variables in the water system. Alternatively or additionally, a disinfection or water control feedback loop can provide a process control signal to the water control system to effect an action on a controllable variable in the water system, for example: increase in UV or ozone treatment; change dosing of disinfectant levels such as chlorine in all its forms; change dosing of peroxide or peracetic acid or other sanitizer levels; adjust pH; adjust water pressure; adjust water flow speed; adjust water temperature; change filtration capacity; cause a cleaning event; add filtration promotion chemicals such as flocculent and coagulant; or other water control measure. In a water system that uses circulating chemical disinfectant, for example, an increase in activity of a disinfectant dosing pump at a particular location can provide the additional chemical disinfection control required to lower the microbiological load. In an example, in-line measurements of hydrogen peroxide demand may call for increasing quantities of disinfectant injections by a dosing pump to maintain a concentration set-point disinfectant value determined to be required to maintain microbiological control. Increasing disinfectant pump speed at particular locations where peroxide sensitive microbiological load has been measured can further provide more granular disinfection treatment at troubled locations in a water system.

[0096] It is notable that chlorine-based disinfectants cannot be used as a proxy for microbiological load in a water system, at least because chlorine has reaction pathways that result in chlorinated organics that are agnostic to whether the organics are from live organisms or dead organisms or organic particulate. As a result, addition of chlorine to a water sample having organic material will change the concentration of chlorine species in solution without correlation to the state of the organic material that it is reacting with. In addition, chlorine has limitedstability in water systems, in particular under high temperature, such as temperatures above 35°C. In a water system where chlorine is used as a disinfectant, the chlorine must be neutralized to measure the microbial load in the water using peroxide. This is because the chlorine affects initial peroxide degradation and also can react with other chemical or organic parameters in the water. To do this, chlorine can be removed from the water sample without interfering with the microbiology in the sample by neutralizing the chlorine and removing it from the background. In one method, removal of chlorine can be done via a calculated concentration of peroxide, using 0.7 ppm of peroxide to neutralize 1 ppm of chlorine. After the chlorine has been removed, peroxide can be dosed into the water sample and degradation of peroxide concentration over time can be measured.

[0097] Figure 3 is a flowchart illustrating a method for measuring microbial load in a water sample. In the method, a known volume of water sample and optionally a dilutant and also optionally one or more additional sample reagents are added to a reaction reservoir to prepare the water sample. Other additional reagents or preparative procedures could include minor pH adjustments of the sample to further remove background colour or turbidity, a membrane filter to remove particulate matter but not microbes, and addition of nutrient solutions to observe and predict possible microbial dominance and outcomes. A known volume of stabilized hydrogen peroxide at a known concentration is added to the prepared sample with a known volume of water. The water sample is then considered to be a prepared sample ready for reaction with stabilized hydrogen peroxide.

[0098] The starting concentration of stabilized hydrogen peroxide reagent in the water sample is preferably between about 2 and 20 ppm, though typically, for most water types, the starting peroxide concentration can be between about 10 and 16 ppm. The reagent value can also be adjusted based on the water quality tested and feedback from the analytical optimization engine on the known or evaluated microbiome. Higher peroxide reagent values are less desirable because, at higher concentrations, the detected synergistic or selectivity effects are diminished. At concentrations of SHP above about 125 ppm, the peroxide in solution may also start to affect any present microbes that are not generally peroxide sensitive at low peroxide concentrations, such as beneficial microbes. The prepared water sample is then allowed time to react or incubate with the hydrogen peroxide. After a delay time, an aliquot of the reacted water sample is 1transferred to a colorimeter along with a known volume of a known concentration of colorimetric reagent (peroxide-reactive dye) and optionally one or more additional reagents.

[0099] The concentration of hydrogen peroxide in the reacted water sample is then measured colorimetrically to provide a colorimetric measurement of hydrogen peroxide demand in the water sample. For measuring the immediate microbial risk in the sample water, a contact time between the peroxide and sample water could be for example between 1 and 60 minutes or between 5 minutes and 60 minutes. Unless a sample has a heavy microbial load, a minimum of 5 minutes is recommended to allow the peroxide to react with the microbiology in the sample water. For immediate microbial risk, the sample time should not exceed 60 minutes, as the maximum reaction time is more than sufficient to detect a possible microbial pathogen signal at low concentrations and make provision for a levelling out due to potentially benefical or competing peroxide resistant microbiology. Measurement of the peroxide concentration in the prepared sample can be done any number of times after a delay time t to obtain a demand curve for the water sample over time.

[0100] Preferably the colorimeter is calibrated with a control sample. In the scenario where the colorimeter cannot be calibrated due to heavy color in the sample water, the water sample can be diluted either before or after incubation with hydrogen peroxide. If after maximum dilution, the system still cannot be calibrated, a matrix solution or diluent can be added and / or the sample can be filtered through a sediment filter (e.g. 0.45 microns) to filter the water sample. The filter pore size does not stop the microbes from going through the membrane but filters out the sediment particulate, which can interrupt colorimetric measurement. If peroxide is being used as a disinfectant in the water process from which the water sample is taken, the MAS testing procedure can be adjusted to account for the presence of a baseline amount of peroxide in the sample water. In this case, the initial amount of peroxide in the sample water can be tested and used to determine MAS scores through a quality control (QC) check to evaluate the residual in the sample before it is sequestered for progressive MAS analysis. For water samples with greater than 1 ppm of peroxide used at baseline, no demand reagent is needed. The minimum contact time can also be adjusted depending on the QC check of the peroxide concentration. The lower the starting concentration, the shorter the contact time. The higher the starting concentration, the longer the contact time. The output results of the colorimetric reaction can then be sent to thewater health processor which will calculate the adjusted MAS. An artificial intelligence or machine learning analytics engine can also be connected to the water health processor and is used to provide actionable intelligence for process control of water quality in the water system based on existing data from the water system and / or from other water systems.

[0101] Figure 4 is a schematic diagram of a system for measuring microbial load in multiple water samples to provide an automated system and apparatus for a MAS sampling procedure. Water samples are either manually collected in a sample bottle and connected to the system or water sample lines can be connected directly to the system. Measurement system 10 shown enables measurement of peroxide demand from multiple water samples at a time in a fluidly connected colorimetric measurement system. Water sample reservoirs 12a, 12b, 12c, which can also be water lines connected directly to the water system, receive water from a water system. A peroxide reagent reservoir 14 stores and can inject an amount of stabilized hydrogen peroxide into mixing reservoirs 24a, 24b, 24c for reaction with the water sample. Sanitizing rinse reservoir 30 fluidly connected to water sample reservoirs 12a, 12b, 12c can inject sanitizing rinse into the water reservoirs for cleaning. As shown, the system comprises multiple controllable valves that can be opened or closed depending on the system state. Dilution reagent reservoir 16 provides a non-reactive diluent as needed to the water sample, such as distilled or reverse osmosis pure water A peroxide dye reservoir 18 stores a colorimetric dye that reacts with peroxide for the colorimetric measurement of the concentration of peroxide in the reacted water sample. Matrix reagent reservoir 20 stores the matrix reagent for use with highly colored water and when the colorimeter will not calibrate to zero or a stable number for background colour. Optional sediment filter 22 can filter sample water prior to addition of sample water to be tested from water sample reservoirs 12a, 12b, 12c into mixing reservoirs 24a, 24b, 24c. Pump 32 and optional flow meter 34 can be placed in-line to control and measure fluid flow, respectively. Colorimeter 26 has a light source and light sensor for measurement of absorbance through a reacted water sample treated with peroxide sensitive dye to measure the concentration of peroxide in the reacted water sample. Water health processor 28 is connected to the colorimeter to receive data from the colorimeter. A control system comprising a control processor with input / output capability and programmable memory is used to control fluid flow through the measurement system.

[0102] At least one programmable logic controller (PLC) controller and multiple controllable valves are used to automate fluid flow and testing procedures in the MAS testing apparatus as shown. The apparatus can perform, for example, a QC check to determine the initial concentration of the peroxide demand reagent, and can also perform a QC check to determine if there are any microbes present in the dilution reagent reservoir 16, the system lines, or any of the other reservoirs. A cleaning cycle can then be triggered that sanitizes and rinses the system or indicates replacement of the dilutant reagent. The apparatus can also comprise one or more fluid handling components, such as one or more fluid pumps, fluid tanks, fluid conduits, valves, and valve manifolds. The system can also comprise one or more control panels, display devices, microcontrollers, solenoids, valves, flow restrictors, temperature control components, pressure control components, drivers, fluid flow components, regulators, chemical sensors (such as for pH, salt concentration conductivity, dissolved oxygen, oxidation-reduction potential (ORP), oxidizability or permanganate index, etc.), physical sensors (such as for temperature, pressure, air flow, fluid flow, turbidity, water clarity etc.), communication and data lines, connection ports, control panel connection banks, and the electrical and / or communication connections there between. The controllable valves can be of any type, including but not limited to, solenoid valves, pinch valves, and proportional valves.

[0103] MAS can be measured by the apparatus using one or more photometric colorimeters, to which will be directed a small amount of water from one of the mixing reservoirs 24a, 24b, 24c along with a peroxide color reagent or peroxide dye, such that a measurement of peroxide concentration in the water from the mixing reservoir after incubation can be done. Mixing reservoirs 24a, 24b, 24c optionally can also comprise a mechanical mixing device to provide homogenous and representative water samples. Measurement of MAS can be repeated multiple times at time intervals for immediate risk analysis and up to, for example, a 20-24 hour period after incubation of the water sample with peroxide in the mixing reservoir (starting at t=0). The MAS resilience can thereby be measured, which observes water resilience over time. During the same cycle, the system can also measure the MAS for the other mixing reservoirs in turn. The collected data is received by water health processor 28 and submitted for computation and analysis to one or more computational engines to enable interpretation of the MAS and adjusted MAS, or MAS-adjusted (MASa) values and provide an overall risk calculation for the watersystem from which the water sample was taken. The water health processor may also prescribe an action based on the risk calculation performed. The action may include adjustment of anyone of a number of controllable variables which may include but are not limited to flowrate, pressure differential, filter operation and backwashing, water level, chemical level, flow rate, pump rate, and water temperature.

[0104] Once the measurements are complete, the mixing reservoirs are emptied, and a new set of measurements can be taken depending on the programmed testing scheduling. The sanitizing reservoir is used to clean the mixing reservoirs 24a, 24b, 24c and the water lines to ensure that no interference microbes are able to grow in those areas. In the scenario where the system cannot calibrate due to heavy color in the sample water, the system can specify a dilution setpoint, based on water clarity and calibration compliance. The system can then reduce the water sample volume input into the mixing reservoir and add an amount of dilution reagent from the dilution reagent reservoir 16 or matrix reagent from the matrix reagent reservoir 20. If after maximum dilution, the system still cannot calibrate, matrix solution can be added from the matrix reagent reservoir 20. There is also the option for the optional sediment filter valve to be opened, into the testing loop, before sample water is directed to the photometric colorimeter. The system can also adjust if peroxide type products are being used in the water treatment process. This can be validated through the QC check. In this case, the time interval for the initial test reading can be adjusted, based on the QC check, as well as the injection amount for the peroxide demand reagent.

[0105] Figure 5 is a schematic diagram of a system for monitoring and controlling microbial load in water. The measurement system 10 is used to determine the MAS, which is a measure of the concentration of peroxide sensitive microbes present in a given water system. The measurement system 10 comprises one or more water sample reservoir, peroxide reagent reservoir, dilution reagent reservoir, peroxide dye reservoir, matrix reagent reservoir, sanitizing rinse reservoir, mixing reservoir, flow meter, pump, colorimeter 26, and sediment filter, as shown in Figure 4. The measurement system 10 also comprises a plurality of fluid flow control devices, such as valves and solenoids, which can be controlled by a programmable logic controller (PLC). The MAS can be calculated by the water health processor 28 based on the change in concentration of stabilized peroxide over time and is indicative of peroxide sensitivemicrobial load. The water health processor 28 also collects data from a number of sensors and controls various controllable variables. The sensors may vary based on the water system being monitored and include but are not limited to a turbidity sensor 42, a pH probe 44, temperature probe 46, conductivity probe 48, dissolved oxygen probe 50, flowmeter 52, water usage sensor 54, water waste sensor 56, pump flow rate and speed sensor 58 and variable frequency drive sensor 60. The sensor and probe readings can be analyzed by the water health processor 28 and proactive responses may be executed based on the results. In one example, the turbidity sensor readings could be compared with the MAS calculations. The water health processor can also receive Al analytics from an Al analytics engine trained on water health data to provide feature analysis of measurements received from the water system. Based on input to the water health processor from the plurality of sensors, and measurement system 10 which provides the calculations of MAS in the system, the water health processor can provide actionable intelligence for water system process control. In one example, a low MAS reading and high turbidity may indicate that there are suspended solids that need to be removed. A proactive response may be to either improve filtration or perform dilution or both.

[0106] An example of a water system that may be monitored and controlled by the system illustrated in Figure 5 is a recreational water system such as a pool or spa. These types of water systems often use chlorine-based disinfection systems which may use sodium hypochlorite for example and may produce mono-, di-, and tri-chloramines when exposed to amines in pool or spa water. These specialized recreational water systems also have controllable variables, monitored by different sensors, that differ from potable and agricultural water systems, such as pool turnover rate, deck temperature, chemical level sensors, filter operation and backwashing and flowrate. In such a water system, the MAS may be determined by the water health processor 28 as exceeding a given threshold value. To bring the MAS to an acceptable value, the water health processor 28 may, using data from a sensor such as a chemical level sensor, increase a controllable variable such as the level of disinfectant added to the water system. This is just one example of how the water health processor may be used to monitor and control the microbial concentration of a water system that uses any type of disinfectant.

[0107] MAS Evaluation of Beneficial Soil Bacteria

[0108] A variety of beneficial soil microorganisms are added to agricultural systems to support plant growth. Beneficial soil bacteria can provide nutrients to plants, secrete protective chemicals, and outcompete deleterious microorganism that may harm the plants or cause poor conditions for plant growth. Some commonly found microbes that are beneficial in soil for agriculture include but are not limited to Lactobacillus Casei, L. Plantarum, Lactobacillus Helveticus, B. subtilus, Bacillus lichenformis, B. pumilos, P. chloroaphis, T. harzianum, Trichoderma virens, Bacillus complex, Trichoderma complex, Lactobacillus complex and other nitrogen fixing and nitrogen converting microorganisms. Evaluation of the MAS effect on these beneficial soil microorganisms can enable a calculation of an adjusted MAS taking into account the effect of these microorganisms on peroxide demand in the MAS calculation.

[0109] P. chlororaphis is a beneficial soil Pseudomonas which is used to inoculate soil in agriculture and horticulture to protect against infection by fungal plant pathogens. Figure 6 is a graph showing the degradation of hydrogen peroxide over time caused by P. chlororaphis at a test sample concentration of 1g P. chlororaphis / L water. A linear trendline of the degradation of peroxide over time was observed as:% = 0.5189% orMASPS= 0.5189t where:MASps is the Microbial Activity Score of the / < chlororaphis [%]; and t is time in minutes.

[0110] The trendline for the MAS of the P. chlororaphis is linear over the time scale t >= 0 minutes and t <= 60 minutes. Therefore, the beneficial effect that P. chlororaphis has on the MAS of a water sample can be taken as equal to the MAS of P. chlororaphis at time t as determined by calculating the contribution of the MAS in the presence of at time t using MASps as set out above. The beneficial effect (BE) that P. chlororaphis has on the MAS of a water sample can be taken as equal to the MAS of P. chlororaphis at time t as:BEpS= 0.5189 /

[0111] Figure 7 is a graph showing the change in the MAS over time caused by Lactobacillus complex at a test sample concentration of 1 million cfu / mL over a 60 minute timerange. To calculate the effects on peroxide, the 1 million cfu / mL Lactobacillus complex test sample was exposed to stabilized hydrogen peroxide and the change in concentration of the hydrogen peroxide was measured over time. The beneficial effect (BE) equation for various inputs for Lactobacillus complex concentration, assuming the effects of Lactobacillus complex on the MAS is linearly scalable for the different concentrations, where:BELB= 0.003t2- 0.2436tThis illustrates that the presence of Lactobacillus complex, which is not a peroxide sensitive microorganism at the peroxide concentrations tested, shows very little effect on the concentration of peroxide in the water sample.

[0112] Figure 8 A is a graph showing the change in MAS over time caused by Lactobacillus complex at a test sample concentration of 10,000 cfu / mL over a 60 minute time range. Taking the average of the two equations in the graphs above (Figures 7 and 8A), the mean equation is found to be:% = 0.003%2— 0.2436% orMASLB= 0.003t2- 0.2436t where:MASLB is the Microbial Activity Score of the Lactobacillus complex [%]; and t is time in minutes.This measurement is valid for MAS Risk tests: t >= 0 minutes and t <= 60 minutes.

[0113] The equation above determines the MAS of the Lactobacillus complex sample over time. Therefore, the beneficial effect (BE) that Lactobacillus complex has on the MAS of a water sample can be taken as equal to the Lactobacillus complex ’s MAS:BElactobacillus= 0.003t2- 0.2436tThe graphs and equations above show that the Lactobacillus complex concentration has very little effect on the BE equation.

[0114] Figure 8B is a graph showing the change in MAS over time caused by Bacillus spectrum over a 60 minute time range. To calculate the effects on peroxide, the lOOpL Bacillus complex test sample was exposed to stabilized hydrogen peroxide and the change in concentration of the hydrogen peroxide was measured over time. The beneficial effect (BE)equation for various inputs for Bacillus complex concentration, assuming the effects of Bacillus complex on the MAS is linearly scalable for the different concentrations, is found to be:MASBS= 0.2808t where:MASLB is the Microbial Activity Score of the Bacillus complex [%]; and t is time in minutes.

[0115] The equation above determines the MAS of the Bacillus complex sample over time. Therefore, the beneficial effect (BE) that Bacillus complex has on the MAS of a water sample can be taken as equal to the Bacillus complex ’s MAS:BEBS= 0.2808t

[0116] Figure 9 is a graph of the change in the MAS over time caused by Trichoderma complex over a 60 minute time range. An experimental solution of Trichoderma complex was created and allowed to grow in solution. The original concentration of Trichoderma complex was 4.25 g in 500 mL (8.5g / L) of distilled water. 3 mL of this solution was then diluted with 1 L distilled water. Two additional samples of differing Trichoderma complex concentrations were tested, and the mean of all three samples and the accompanying standard deviations are included in Figure 8. The mean equation is found to be:% = 0.0833% + 2.4066 orMASTR= 0.0833t + 2.4066 where:MASTR is the Microbial Activity Score of the Trichoderma complex [%]; and t is time in minutes.

[0117] The low standard deviations show that the concentration of Trichoderma complex has little effect on the BE equation. This trend has been found to be valid for MAS risk tests for Trichoderma complex between MAS time points t >= 0 minutes and t <= 60 minutes. Therefore, the BE that Trichoderma complex has on the MAS of a water sample can be taken as equal to the Trichoderma complex MAS as follows:BETR= 0.0833t + 2.4066This illustrates that the presence of Trichoderma, which is not a peroxide sensitive microorganism at the peroxide concentrations tested, shows very little effect on the concentration of peroxide in the water sample.

[0118] Effect of Metals on MAS

[0119] Figure 10 is a graph illustrating the effect of iron on the degradation of hydrogen peroxide at various iron concentrations over time. This graph shows the effect that metals, specifically iron, have on the MAS reading or peroxide concentration over time and illustrates the peroxide concentration degradation at varying concentrations of iron and peroxide. Iron reacts with hydrogen peroxide and can be used as a proxy for possible metal interference in MAS calculations. The mean MAS of iron (Fe) samples containing chelated iron and total iron as Fe2+and Fe3+were tested in the matrix solution over time at a concentration of around 1 ppm iron.The effect of iron on the MAS score is shown below, which is approximated to be constant based on the data. Equations listed below:Iron Sample 1 (Iron 1) MAS: MASlronl = 0.0257 ItIron Sample 2 (Iron 2) MAS: MASlron2 = 0.01529tMean Iron MAS: MASi ron,mean 0.02t where:MASiron = Microbial Activity Score Interference of the Iron [%]; and t = time [minutes],

[0120] The Metals Effect (ME) that iron has on the MAS of a water sample can be taken as equal to the iron’s mean MAS:MEIron= 0.02tAssuming all iron-type metals will behave similarly to iron with respect to their effect on the MAS, the concentration-dependent effect ME on MAS is as MEpe= 0.02t. The ME equation can then be generalized for various user inputs for iron concentrations. It is also observed that the effects of iron on the MAS is linearly scalable for the different concentrations.

[0121] Effect of Total Organic Carbon on MAS

[0122] Figure 11 is a graph showing the effect of total organic carbon (TOC) on the degradation of hydrogen peroxide over time in a water sample. In particular, the effect of theTotal Organic Carbon (TOC) on peroxide degradation was investigated. A water sample was tested for TOC before and after peroxide exposure to see if anything happened to the TOC level or the peroxide concentration. It was observed that the TOC level did not decrease and that the peroxide concentration remained stable. The TOC level tested in the experiment was average for untreated water, though concentrations between 2 and 25 ppm have been observed in various surface and process water samples with little effect on the MAS calculation.

[0123] Figure 12 is a graph of a multiple-component factor effect on the degradation of hydrogen peroxide over time. Water in different water systems can have a wide variety of physical and chemical components, such as dissolved salts and metals, range of pH, among other varying factors tested. A plurality of the most important and re-occurring factors were tested that are commonly found in treated water systems and could potentially have an effect on stabilized peroxide degradation over time. In particular, water systems vary in alkalinity, pH, temperature, and metal salt concentration, among other factors. Table 1 provides a sample control composition comprising a number of commonly found components in treated water which was used for this investigation.Table 1 :

[0124] Based on the slope of the graph in Figure 12, the multiple component factor effect on MAS over a time scale of t >= 0 minutes and t <= 60 minutes is shown to be 0.0182t. As such, it can be concluded that the components in the control sample water do not have a substantial effect on the degradation of stabilized hydrogen peroxide in water over a 0-60 minute time course. As such, it can be concluded that normal variations in the control factors tested do not significantly affect the calculation of MAS for the water sample.

[0125] Sanitizer Effect On MAS

[0126] The presence of chlorine-type sanitizers will have an effect on the concentration of peroxide demand reagent, causing a decrease in the demand reagent concentration and, as a result, an increase in the MAS value. The Sanitizer Effect (SE) value will account for this in the Microbial Activity Score - adjusted (MASa) calculation (see Adjusted MASa section below). The effect chlorine has on the concentration of stabilized hydrogen peroxide reagent in the water sample is linear based on the concentration of chlorine. For every Ippm of chlorine present in the sample, the peroxide demand reagent concentration will decrease by 0.65 ppm. To determine the effect of chlorine on the MASa calculation, the MAS value of chlorine can be found as follows using the MAS equation: 100Knowing that the effect of chlorine on peroxide is a linear reduction based on the chlorine concentration, the equation becomes: 100This equation is then reduced to:1.538 x CL100which can also be written as:1.538 x CclSE x 100% Demand.QC (:heckwhere:SE is the Sanitizer Effect [%];Cci is the Concentration of chlorine-type chemicals present [ppm]; andDemandqc check is the quality control (QC) Check Demand Reagent concentration in ppm.

[0127] In a water system comprising a chlorine based disinfectant, the calculation of MAS can still be done by first reacting any residual chlorine species with extra hydrogen peroxide. To do this, an additional 0.65 ppm of stabilized hydrogen peroxide can be added to the water samplefor every 1 ppm of chlorine in the water sample to remove the chlorine from the water sample before calculating peroxide concentration degradation in the water sample.

[0128] Adjusted MAS (MASa)

[0129] An adjusted MAS (MASa) can be calculated by taking into account the MAS of a water sample and further including the presence of other contributing factors. Microbial ActivityScore Adjusted, or MASa, is determined to account for the effects of the beneficials or peroxide non-sensitive microbes and metals on the MAS, in this case MAS Risk:

[0130] Some inputs to the adjusted MAS calculation can include but are not limited to: quality control reagent check; MAS risk (short); MAS resilience (long); reaction time; dilution; concentration of Pseudomonas-type organisms; concentration of Bacillus-type organisms; concentration of Lactobacillus-type organisms; concentration of Trichoderma-type organisms; concentration of iron or iron-type metals; concentration of total organic carbon (TOC); alkalinity; calcium hardness; concentration of one or more species of chlorine-type sanitizers; conductivity; total dissolved solids (TDS); pH; turbidity; and water system temperature. MASa can be used during analysis, and MASa computation can provide actionable intelligence to determine the microbial risk based on water measurement data, including the peroxide demand as measured by the colorimeter. In some examples, a ‘short’ MAS calculation is used for times for reaction times between 0-60 minutes after peroxide addition, such as between 1-60 minutes, and the Tong’ MAS calculation is used for times for reaction times longer than 60 minutes after peroxide addition, such as between 60 minutes and 24 hours. The analytics engine implements machine learning models which include data integration, pattern recognition, predictive risk analysis, and deep learning, which provide predictive and prescriptive water process optimization feedback to maintain microbiological control in the water system. Multiple linear regression analyses can be utilized to evaluate the linear relationship between the multiple MAS factor inputs, fit linear equations to predict microbial risk and future trends, provide comprehensive insights into the underlying data dynamics. Concurrently, machine learning optimization methodologies have been implemented, allowing for the integration of intelligent feedback mechanisms. Machine learning optimization takes in all appropriate MASa data,degradation trends, and previous feedback, and enhances the predictive accuracy, process evaluation, and provides valuable improvement-focused feedback loops on disinfectant levels and microbiome balance. Optimization also increases computational efficiency and accuracy of the machine learning systems, thereby enabling them to better identify real-world root cause analysis, recommend remediative action, and make informed decisions.

[0131] MAS Testing in a Mixed Microbiome

[0132] Figure 13 is a graph showing the effect of Lactobacillus on MAS vs time in the presence of E. coli. The experiment included both moderately deleterious peroxide sensitive E.coli and beneficial peroxide non sensitive Lactobacillus in a mixed process water sample. The initial peroxide demand reagent value decreased rapidly and then tapered off. This mixed bacteriological culture demonstrated a significant reduction in concentration of potential pathogen (E.coli) but growth in present Lactobacillus, which was also visible in the reagent value over time. This supports the mechanism of action for Lactobacillus as being non sensitive to peroxide and the potential usefulness of the test in a mixed microbiome to distinguish between peroxide sensitive microbes and non peroxide sensitive microbes. The mean equation for the MAS is found to be:MASLBIE= 0.0002t3- 0.0546t2+ 3.3241t where:MASLB.E is the Microbial Activity Score of the Lactobacillus in the presence of E. coli [%]; and t = time [minutes] for MAS Risk tests, where t >= 0 minutes and t <= 60 minutes. Over time, the mean for the MASa can be approximated. A reduction in concentration of pathogenic E. coli is also evident.

[0133] Figure 14 is a graph showing the measurement of MAS for a mixed sample containing Trichoderma complex and coliforms over a one-hour time course. As observed, the MAS increases quickly until the 15-minute mark, with this increase likely due to the presence of coliforms in the water, increasing up to 46%. At this 15-minute mark, Trichoderma seems to compete to outperform the coliforms, possibly eliciting its own mechanism of action, resulting in the MAS improving slightly. As the demand initially goes up (peroxide reagent value decreases), then slightly comes down (or reagent value increases slightly, after which is remains stable forthe long demand period. This improvement continues until the 30-minute mark, where the slope begins to level at a MAS of approximately 29.70%. This sample was sent off to the laboratory for verification, which confirmed that the sample was replete with Trichoderma and no coliforms were detected. The concentration of circulating cellular ATP (cATP) was tested over the same time course, with the concentration of cATP at t=0 at 4845 pg / mL and the concentration of cATP at t=60 at 4861 pg / mL. This indicates that cATP method, though able to provide an overall picture of microbiology in a water sample, is ineffective in distinguishing between beneficial peroxide non sensitive Trichoderma microbes and the peroxide sensitive coliforms in the water sample. In the tested mixed microbiome, there are sufficient beneficials (e.g. Trichoderma') present to outperform the pathogen (coliforms versus a water sample where the coliforms dominate, as illustrated in Figure 14.

[0134] Figure 15 is a graph showing the measurement of MAS for a sample containing only coliforms over 60 minutes. The baseline coliform concentration was 1345.69pg / ml. As observed, the MAS increases quickly such that by 30 minutes there is no peroxide remaining in the water sample. In the absence of a competing beneficial microbe such as Trichoderma, the peroxide demand reagent is high and used up rapidly with only 4.5ppm at t=0. The MAS of the coliforms sample continues to increase after the 15 -minute mark, up to the maximum of 100% by the 30- minute mark. The concentration of circulating cellular ATP (cATP) was tested over the same time course, with the concentration of cATP at t=l at 738 pg / mL and the concentration of cATP at t=60 at 741 pg / mL.

[0135] A MAS computation can provide valuable insight into what is happening in the water between the peroxide non sensitive beneficial microorganisms and possible pathogens that are sensitive to peroxide such as coliforms. As shown, the cATP method cannot provide this insight as it can only measure the quantity of all living organisms in the water. The MAS data, graphs and correlation trends are fed into a tailored machine learning analytical engine which is used to provide valuable insight and predictive analysis into real-time practical water health applications. The machine learning model can be used for pattern recognition, predictive risk analysis, deep learning for graphs, and data integration, as well as providing process optimization outputs for improving the water treatment processes, specifically related to microbiome resilience, disinfection efficacy, and mitigation of disinfection by-products.

[0136] Example 1: Greenhouse ApplicationIn a greenhouse water circulation system, MAS was sampled in a short time interval and a long time interval periodically over the course of two months. Table 2 illustrates the MAS reporting for first greenhouse location, and Table 3 illustrates the MAS reporting for the same greenhouse in a re-use section.Table 2:Table 3:

[0137] Figure 16 is a graph illustrating microbial risk levels compared with water temperature in a greenhouse water circulation system in two different irrigation tanks. Microbial risk detection and remediation by temperature adjustments in the presence of beneficial P. chlororaphis possibly negated the effect of P. chlororaphis on the MAS. By sampling the MAS and temperature of the water, the present system provided insight into the operation of the system, ensuring that temperature adjustment by means of a chiller and addition of beneficial P. chlororaphis to the water system would improve the MAS score. Other examples of benefits observed in greenhouse water systems include providing an indication that preventative maintenance and cleaning of the greenhouse water circulation system is required to prevent overgrowth, reducing the volume of the waste stream, reducing energy use, and increasing the useful life of the water infrastructure. Optimization of the health of incoming water into agreenhouse facility where the on-line MAS testing was used to drive pH and sanitizer control was also found help to ensure optimal disinfection of incoming water. The outcome of this test facility saw a marked reduction in disinfectant use over the same period the previous year, despite increasing the size of the greenhouse.

[0138] MAS sampling and calculations have been used in other greenhouse facilities to identify local disease pressures in the system, depending on the point of use zone in the greenhouse. In particular, during one investigation, one bay in a greenhouse zone was identified as having a much higher MAS than the other bays in the same zone with all other factors being equal. This local contamination in the section of pipe which supplies water to the infected bay was found to be caused by mechanical debris inside the pipe that was serving as a growth surface for deleterious microbes. Identification of this high peroxide demand area in the greenhouse circulating water system triggered maintenance to the piping in the infected bay. Once cleaned, the MAS values returned to normal, in line with the rest of the greenhouse zones, after the preventative action was carried out.

[0139] In another greenhouse test facility filtration was being used to manage the organic content in recycled water tanks. Organic load in the filter led to additional chemical consumption in the filtered water due to microbial nutrients being made available. Conventional backwash indicators did not pick up on this early as these only look at pressure build up in the filter tanks as an indication of required maintenance. MAS testing before and after the filters provided early visibility on microbial loads increasing in the filtration system and were able to guide the backwash frequency and duration, facilitating better water quality in the tanks and downstream and reduced disinfectant use for water sanitization.

[0140] It is noted that depending on the quality of the water to be sampled, the demand reagent level can be adjusted. High microbial load process water will elicit a better test result and provide more insight with a starting concentration of SHP of at least 15ppm with high nutrient water, other details can be considered to provide revised settings in the system to deal with the initial demand. Ranges of between about 1 and 30 ppm of concentration of SHP at the start time in the water sample are preferable, and have been found to provide reliable test results. More preferably, the starting concentration of SHP in the water sample for the demand test is 2-20 ppm. Dilution factors can also be adjusted for dark water to facilitate a successful colorimetrytest. In some examples a dilution factor can be applied, such as 1 : 1, 1:2, 1 :5, or 1 :10, to dilute the water sample. It is also understood that above a certain peroxide concentration, for example above about 125 ppm, the peroxide will indiscriminately oxidize all oxidizable microbes that may be non-reactive to peroxide at test concentrations according to the present method, and that all microbes are susceptible to high peroxide concentrations.

[0141] Where the demand test shows rapid degradation, preventative action needs to be taken to remediate the unusual demand or upward trend. Continuous testing systems such as one or more colorimeter with in-line units can conduct this demand testing and other monitoring on an ongoing basis and can do so with continuous dosing of disinfectant or other inputs such as beneficials to keep the process water in good microbiological control. In a potable water system a demand test or test series should be conducted at least daily, and more preferably multiple times daily, to ensure proper water monitoring. Where the demand test shows rapid degradation, preventative action needs to be taken to remediate the unusual demand or upward trend. Rapid degradation of demand reagent in the test sample, or the SHP level already in a water system, indicates that the MAS and contamination risk is high. In one example the MAS can be expressed as a % MAS, with risk reported, in one example, as a percentage over time as follows:

[0142] Figure 17 illustrates results from a graphical user interface of a water health index system reporting health insights based on MAS. MAS resilience testing between 20-24 hours looks at microbial background and buffer capacity of a water sample and which microbes dominate. The adjusted MASa resilience for long demand may be adapted if beneficials are being used in the water system of the sampled water. In one example, a value of less than 70 indicates that the MASa is on track, greater than or equal to 90.01 and less than or equal to a MASa of 95 indicates caution, and greater than 95.01 indicates an at risk condition. Valuesbetween 70 and 90 can indicate different conditions based on the type of water and prior testing. In the absence of a MASa reporting, the water status can be set at a default status of “at risk”.

[0143] Figure 18 is a graph of iron vs. concentration of stabilized hydrogen peroxide in solution. Shown is the analyzed interference of 1 ppm iron ethylene diamine tetra-acetic acid (EDTA) solution in relation to microbial activity score (MAS) for two samples. Adjusted MAS or MASa further adjusts the calculation in the computational engine to make provision for an adjusted MAS percentage and possible risk reduction or augmentation, based on the different contributing effects, such as MMFE, BE, and ME. In the case of Figure 17, the ME will be accounted for in the MASa calculation to appropriately adjust the MAS percentage as iron is present in the sample water. An automated monitoring system will provide feedback and can query the water when needed, including the initial MAS and multiple MAS calculations over time (minutes / hours / days). Rapid degradation with a MAS greater than 30% indicates that the MAS and microbial contamination risk is moderate. High risk is observed when the demand reagent or SHP concentration is substantially degraded over the period of time measured, which can be observed as a MAS of 60-100%, with the highest risk as the score progresses closer to 100%.

[0144] Example 2: Hospital Shower Hotspot

[0145] MAS risk was identified during the test for a hospital shower hotspot that showed a microbial risk signal. Figure 19A illustrates a graphical user interface reporting a high risk condition for a hospital shower using a MASa short quality control (QC) check. Figure 19B illustrates the MAS data showing the MASa (short) data over time compared to cATP data over time indicating a microbial hot spot at the shower. Review of MAS short scores over a number of months at the same hotspot compared to cATP. MAS risk signal was moderately high in January for shower hotspot with 59.38% as confirmed by cATP at 15.05 pg / ml. The shower was preventatively remediated with shock disinfection of the fixture and flushing. Post-remediation MAS Short values showed moderately low risk as also confirmed by cATP sampling at 0.96pg / ml. It is notable that cATP showed an increase much later than the observed increasing MAS score based on peroxide demand. Accordingly, using MAS to calculate microbial risk can be used as an early indicator of increasing peroxide sensitive microbe concentration. In Figure 19B, the MASa reading on Nov 15th (2023) shows increased microbial risk in the shower, whichis not shown by the cATP readings at that time. Later on Jan 5th (2024), the cATP finally reflects the higher MASa score. Based on this, MASa is a more effective early indicator for increasing microbial activity and risk.

[0146] Example 3: Propagation Greenhouse

[0147] Figure 20 illustrates MAS data of a water tank in a propagation greenhouse. An increase in turbidity had a strong correlation with rapid increase in MAS short with 100% being the percentage over a 15 minute MAS test. This lead to preventive cleaning of the tank, eliminating the risk and substantially reducing the turbidity in the re-use water tank. Turbidity may be related to inert particulate in the water but may also be related to the microbial load. The MAS test was used to determine risk and prevent a pathegenic pressure building up in the system.

[0148] Figure 21A illustrates a graphical user interface after detection of high MAS and before remediation in the water tank. Figure 2 IB illustrates a graphical user interface after detection of high MAS and after remediation of the tank.

[0149] Example 4: Greenhouse Secondary Treatment

[0150] Figure 22 illustrates MAS data in a greenhouse silo. The greenhouse used SHP for secondary disinfection treatment. MAS tests showed 0% long MAS scores and MAS short scores were used to detect a reduction in resilience in Silo 2, as shown. Further evidence of infection was observed by a subsequent slight increase of yeast in tank 2 (22 cfu / ml). Remediation was conducted through the addition of beneficials and a slight increase of SHP disinfectant levels to the water system.

[0151] Example 5: Greenhouse Secondary Treatment

[0152] Figure 23 illustrates MAS data at a tray seeder table in a greenhouse. Microbial risk detection and resilience improvement in trays for seedlings in this greenhouse application were remediated by sanitizer. A number of MAS short demand tests at 15 minutes were done, as well as a MAS long tests between 22 and 24 hours. When the first MAS short spike was detected, sanitizer levels were increased, which led to reduced MAS Risk results. Beneficial microbes were added to the water system as MAS resilience showed greater demand, which subsequently improved MAS scores.

[0153] Figures 24A-E illustrate the present system in a benchtop device, where: Figure 24A illustrates a front isometric view of the present system in a benchtop apparatus; Figure 24B illustrates a rear isometric view of the present system in a benchtop apparatus; Figure 24C illustrates a top view of the present system in a benchtop apparatus; Figure 24D illustrates a front isometric view of the present system in a benchtop apparatus with an open door or removable panel; and Figure 24E illustrates a front isometric view of the present system in a benchtop apparatus with a removable or sliding front panel showing the mixing reservoirs. The benchtop apparatus or device can be used as a benchtop lab, or can be connected to a water system for periodic or continuous monitoring of water in a water system. The benchtop system includes a colorimeter having one or more measurement cells, sensor probes, reagent reservoirs, dilution and aliquot vessels, and a data hub with a communication module for input and output data, a processor or Programmable Logic Controller (PLC), digital display, and a control panel. The benchtop apparatus can also have one or more communication ports, data ports, input / output ports, wireless communication devices, and memory devices.

[0154] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains and are herein incorporated by reference. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that such prior art forms part of the common general knowledge.

[0155] The invention being thus described, it will be obvious that the same may be varied in many ways. Such variations are not to be regarded as a departure from the scope of the invention, and all such modifications as would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims

CLAIMS:

1. A method for detecting hydrogen peroxide sensitive microbes in a water system comprising: obtaining a water sample from the water system; adding an initial known amount of stabilized hydrogen peroxide (SHP) to the water sample to provide an initial concentration of hydrogen peroxide in the water sample; waiting a delay time; and calculating a microbial activity of peroxide-sensitive microbes in the water sample by colorimetrically determining a concentration of hydrogen peroxide after the delay time.

2. The method of claim 1, wherein the stabilized hydrogen peroxide is hydrogen peroxide stabilized with silver.

3. The method of claim 2, wherein the silver comprises one or more of silver ions, oligodynamic silver, silver nanoparticles, and silver colloid.

4. The method of any one of claims 1-3, wherein the initial amount of stabilized hydrogen peroxide provides a hydrogen peroxide concentration in the water sample of between about 1- 125 ppm.

5. The method of any one of claims 1-4, wherein the water sample is taken at multiple locations in the water system.

6. The method of any one of claims 1-5, wherein the water system is a potable water system, agricultural water system, horticultural water system, aquaculture system, or recreational water system.

7. The method of any one of claims 1-6, wherein the microbial activity score (MAS) in the water system based on the hydrogen peroxide degradation rate over time is calculated as:where [SHP] is the concentration of stabilized hydrogen peroxide in the water sample.

8. The method of any one of claims 1-7, wherein the calculation of the MAS further comprises adjusting the MAS calculation based on one or more of quality control reagent check, MAS risk (short), MAS resilience (long), reaction time, dilution, concentration of Pseudomonas- type organisms, concentration of Bacillus-type organisms, concentration of Lactobacillus- ypeorganisms, concentration of Trichoderma- Q organisms, concentration of iron or iron-type metals, concentration of total organic carbon (TOC), alkalinity, calcium hardness, concentration of one or more species of chlorine-type sanitizers, conductivity, total dissolved solids (TDS), pH, turbidity, and water system temperature.

9. The method of any one of claims 1-8, further comprising adjusting a controllable variable of the water system when the microbial activity score of peroxide sensitive microbes in the water sample is greater than a safety threshold for microbial control.

10. The method of claim 9, wherein adjusting the controllable variable comprises one or more of dosing the water system with a chemical disinfectant, increasing the pump rate of a disinfectant pump into the water system, increasing ultraviolet treatment in the water system, shocking the water system with a chemical disinfectant, increasing ozone production in the water system, adding beneficial microbes to the water system, and performing a maintenance operation on the water system.

11. The method of claim 9 or 10, wherein adjusting the controllable variable lowers the microbial load of peroxide sensitive microbes in the water system to within the safety threshold.

12. A system for detecting hydrogen peroxide sensitive microbes in a water system comprising: a water sample supply for receiving a water sample from the water system; a peroxide reagent reservoir comprising stabilized hydrogen peroxide; a mixing reservoir fluidly connected to the water sample supply and the peroxide reagent reservoir for receiving and mixing a water sample and stabilized hydrogen peroxide to provide a reacted water sample; a peroxide dye reservoir comprising peroxide dye; a colorimeter for receiving the peroxide dye and the reacted water sample and performing a colorimetric measurement to determine a concentration of hydrogen peroxide in the reacted water sample; and a water health processor for receiving colorimetric measurements from the colorimeter and determining a microbial activity score for the water system based on the colorimetric measurements.

13. The system of claim 12, wherein the system further comprises one or more sediment filter, dilution reagent reservoir, pump, flow meter, sanitizing rinse reservoir, mechanical mixing device, and matrix reagent reservoir.

14. The system of claim 12 or 13, further comprising more than one water sample reservoir and more than one mixing reservoir.

15. The system of any one of claims 12-14, wherein the stabilized hydrogen peroxide is silver-stabilized hydrogen peroxide.

16. The system of any one of claims 12-15, wherein the water sample supply is in line in a water system.

17. The system of any one of claims 12-16, wherein the water health processor is connected to one or more water system sensor selected from the group consisting of a turbidity sensor, pH probe, temperature probe, conductivity probe, dissolved oxygen probe, flowmeter, water usage sensor, water waste sensor, pump flow rate sensor, pump speed sensor, and pump variable frequency drive sensor.

18. The system of any one of claims 12-17, wherein the water health processor further comprises one or more of an artificial intelligence engine and machine learning analytics engine.

19. The system of any one of claims 12-18, wherein the water health processor provides a process control signal to effect an action on a controllable variable in the water system.

20. The system of claim 19, wherein the controllable variable comprises flowrate, pressure differential, filter operation and backwashing, water level, chemical level, flow rate, pump rate, and water temperature.

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